Early neuron-neuron interactions play a crucial role in the localization of postsynaptic receptors and the development and plasticity of functional synaptic connections. Synapses of hippocampal neurons have several characteristics which add complexity to the understanding of the early events in synaptogenesis: (1) synaptic connections may be inhibitory, utilizing the transmitter gamma-amino-butyric acid (GABA), or excitatory, utilizing excitatory amino acids (EAA; primarily glutamate); (2) postsynaptically, at least two receptors subtypes are co- localized for both GABAergic and glutamatergic synapses. The proposed work will answer specific questions concerning cellular and molecular mechanisms of transmitter secretion and postsynaptic receptor localization during early synaptogenesis of hippocampal neurons in culture. Using excised patches of excitable membrane or adult dissociated hippocampal neurons as probes to detect spontaneous neurotransmitter release from growing hippocampal neurons in culture, experiments proposed in part one will characterize the phenotype, distribution, and time course of spontaneous and evoked neurotransmitter (GABA vs glutamate) release of hippocampal neurons in culture. Experiments proposed in part two are aimed at understanding the time course and possible mechanisms of localization/co-localization of postsynaptic receptors, either GABA or EAA receptors. In addition to using iontophoretic mapping techniques, immunocytochemistry and electron microscopy will be used to characterize receptor phenotype and clustering on dendritic, axonal and somatic compartments of developing neurons at different time points before and during synaptogenesis. The results of the proposed experiments will provide the basis for future studies aimed at understanding plasticity of developing synaptic connections of hippocampal neurons. The long-term goal of the proposed and future studies is to understand the time course and sequence of cellular and molecular events involved in central synapse formation and plasticity. An understanding of the formation of synaptic connections in growing hippocampal neurons will provide insights into the necessary components involved in synaptic organization and plasticity of the central nervous system, which are crucial for elucidating neural substrates of both normal and pathological states.